The present study reports the feasibility and successful production of rabbit cG-CAOMECS, designed to reconstruct corneal epithelium of patients with bilateral limbal stem cell deficiency. To produce a safe, chemically defined and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP-certified cell culture surface coated with GMP-grade extracellular matrix. A newly designed clinical-grade medium (KaFa™ medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony-forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells expanded to form a multilayered epithelial cell sheet that was harvested after 17-19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 was expressed in the basal cells and K3/K12 was expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, E-cadherin, beta-catenin, and Cnx43 were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of integrin-beta1 and beta4 confirmed that the collagenase treatment used for detaching and harvesting the cell sheet did not have adverse effects. Our results showed that the utilization of clinical-grade and FDA-approved reagents successfully supported the production of cG-CAMECS.
Keywords: Collagenase treatment; Oral mucosal epithelial stem cells; cG-CAMECS.
© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.